Journal: Pulmonary Circulation

Article Title: The ARP 2/3 complex mediates endothelial barrier function and recovery

doi: 10.1086/690307

Figure Lengend Snippet: Effect of Arp 2/3 inhibition on EC barrier function and recovery. HPAECs or HLMVECs were grown on gold electrodes to obtain measurements of TER. Depicted plots are mean normalized resistance values with standard error from 2–4 repeated measures in each condition. (a, b) Representative normalized TER vs. time plot in HPAECs or HLMVECs subjected to CK-666 (50 µM) or vehicle (0.3% DMSO). (c, d) Quantification of mean baseline resistance values (ohms) 1 h after vehicle or CK-666 in HPAEC or HLMVEC. (e) Representative TER vs. time plot of HPAECs obtained after pretreatment with CK-666 or vehicle followed by S1P (1 µM). (f) Maximal change in normalized resistance following S1P. (g) Maximal rate of change in normalized resistance per hour following S1P. (h) Representative TER vs. time plot of HPAECs obtained after pretreatment with CK-666 or vehicle followed by thrombin (1 U/mL). (i) Mean time to recovery of baseline barrier function following thrombin treatment. Quantified values were obtained by pooling 7–9 independent experiments each with 2–4 repeats of each condition (* P < 0.05 vs. vehicle, paired t-test).

Article Snippet: As indicated, cells were also treated with the monoclonal anti-cortactin 4F11 Alexa Fluor 555 conjugate antibody (EMD Millipore) and/or polyclonal anti-Arp 2 antibody (Cell Signaling) overnight followed by secondary anti-rabbit Alexa Fluor 488 (Invitrogen).

Techniques: Inhibition

Journal: Pulmonary Circulation

Article Title: The ARP 2/3 complex mediates endothelial barrier function and recovery

doi: 10.1086/690307

Figure Lengend Snippet: EC gap formation is increased after Arp 2/3 inhibition. HPAECs or HLMVECs were grown to confluence on biotinylated gelatin. After 1 h pretreatment with CK-666 (50 µM) or vehicle, FITC-avidin was added to media and allowed to permeate cells to reach biotin substrate at sites of paracellular gaps. Shown are representative IF microscopy of interendothelial gaps (green fluorescence) between (a) HPAECs and (c) HLMVECs obtained at baseline and after S1P (1 µM × 15 min) or thrombin (1 U/mL × 30 min). (b, d) Quantification of gap area per imaged field in HPAECs and HLMVECs. Bar graphs represent mean values with standard error from n = 4 (HPAEC) and n = 2 (HLMVEC) independent experiments with 2–3 images quantified per condition. (α = P < 0.05 vs. baseline vehicle, * P < 0.05 between groups, by two-way ANOVA with Sidak’s multiple comparisons test).

Article Snippet: As indicated, cells were also treated with the monoclonal anti-cortactin 4F11 Alexa Fluor 555 conjugate antibody (EMD Millipore) and/or polyclonal anti-Arp 2 antibody (Cell Signaling) overnight followed by secondary anti-rabbit Alexa Fluor 488 (Invitrogen).

Techniques: Inhibition, Avidin-Biotin Assay, Microscopy, Fluorescence

Journal: Pulmonary Circulation

Article Title: The ARP 2/3 complex mediates endothelial barrier function and recovery

doi: 10.1086/690307

Figure Lengend Snippet: S1P induced peripheral actin structures and regulatory protein interactions are altered by CK-666. HLMVECs were treated with CK-666 (50 µM × 1) or vehicle. Cells were fixed, stained, and confocal IF images were obtained at baseline and at 2, 5, 15, and 30 min after S1P (1 µM). (a) Representative images of vehicle (top row) or CK-666 (bottom row) treated cells after staining for cortactin (green), actin (red), and nuclear structure (DAPI, blue). Robust lamellipodia formation with membrane ruffling (white arrows) and associated colocalization of actin and cortactin (yellow) is observed rapidly (2–5 min) after S1P. (b) Confocal IF images of HLMVEC stained for Arp 2 (green), cortactin (red), and actin (blue). Colocalization of all three proteins appears white while cortactin-actin colocalization appears purple. Representative images of vehicle (top row) or CK-666 (bottom row) treated cells are shown. Scale bar = 10 µm.

Article Snippet: As indicated, cells were also treated with the monoclonal anti-cortactin 4F11 Alexa Fluor 555 conjugate antibody (EMD Millipore) and/or polyclonal anti-Arp 2 antibody (Cell Signaling) overnight followed by secondary anti-rabbit Alexa Fluor 488 (Invitrogen).

Techniques: Staining

Journal: Pulmonary Circulation

Article Title: The ARP 2/3 complex mediates endothelial barrier function and recovery

doi: 10.1086/690307

Figure Lengend Snippet: Arp 2/3 inhibition reduces lamellipodia formation and EC contacts during thrombin recovery. Confluent HLMVECs were exposed to CK-666 (50 µM × 1 h) or vehicle and confocal IF microscopy images were obtained 30, 45, and 60 min following thrombin (1 U/mL). Cells were fixed and stained for cortactin (green), actin (red), and nuclear structure (DAPI, blue). Representative images from three independent experiments are shown (6–8 cells imaged per experiment). Cortactin-enriched lamellipodia (white arrows) are observed at 30 and 45 min in vehicle treated cells (top panel) but mostly absent in CK-666 treated cells at the same time points (bottom panel). At 60 min after thrombin, punctate colocalization of actin and cortactin are observed at sites of intercellular contact (yellow ovals) while Arp 2/3 inhibited cells demonstrate persistent stress fibers and intercellular gaps (white stars). Scale bar = 10 µm.

Article Snippet: As indicated, cells were also treated with the monoclonal anti-cortactin 4F11 Alexa Fluor 555 conjugate antibody (EMD Millipore) and/or polyclonal anti-Arp 2 antibody (Cell Signaling) overnight followed by secondary anti-rabbit Alexa Fluor 488 (Invitrogen).

Techniques: Inhibition, Microscopy, Staining

Journal: Pulmonary Circulation

Article Title: The ARP 2/3 complex mediates endothelial barrier function and recovery

doi: 10.1086/690307

Figure Lengend Snippet: Arp 2/3 inhibition reduces lamellipodial depth. Subconfluent HLMVECs were exposed to CK-666 (50 µM × 1 h) or vehicle followed by S1P (1 µM). (a) Representative high resolution (pixel size 15 nm) confocal images of phalloidin stained peripheral actin in vehicle (top row) or CK-666 (bottom row) treated cells at baseline, 2 min, and 30 min following S1P. Individual lamellipodia were identified and outlined. The distance from membrane leading edge to central border was measured at several points (yellow lines). Scale bar = 1 µm. (b) Quantification of mean lamellipodia depth at baseline and following S1P. n = 8–15 cells per group with 2–3 lamellipodia measured per cell. (* P < 0.05 vs. vehicle, by two-way ANOVA with Sidak’s multiple comparisons test).

Article Snippet: As indicated, cells were also treated with the monoclonal anti-cortactin 4F11 Alexa Fluor 555 conjugate antibody (EMD Millipore) and/or polyclonal anti-Arp 2 antibody (Cell Signaling) overnight followed by secondary anti-rabbit Alexa Fluor 488 (Invitrogen).

Techniques: Inhibition, Staining